The present invention is in the field of enzymatic cleavage (deacylation), especially one-step cleavage of the 7-aminoadipoyl side chain (also referred to as 7-.alpha.-aminoadipyl) of cephalosporin C. Since the 7-aminoadipoyl side chain is removed by cleavege of an amide linkage, the particular enzyme which accomplishes the conversion is referred to herein as an amidase. Cephalosporin C itself is a fermentation product which is the starting point for nearly all currently marketed cephalosporins. However, synthetic manipulation to produce these various commercial cephalosporins basically starts with the 7-aminocephalosporanic acid, which must be derived from the cephalosporin C by cleavage of the 7-aminoadipoyl side chain.
Currently, the method of choice in the art for cleaving the 7-aminoadipoyl side chain is chemical. the basic imino-halide process requires blocking of the amino and carboxyl groups on the 7-aminoadipoyls side chain, and several methods for accomplishing this are currently used. However, as presently employed, the chemical cleavage process has serious disadvantages. Among these are the requirements of a multi-step and complex process, extremely low operating temperatures, expensive reagents, significant quantities of process by-products resulting in effluent treatment problems, and purification of a highly impure starting material before chemical treatment begins. Consequently, there has been an ongoing search for a microbiological or fermentative process which would achieve enzymatic deacylation of cephalosporin C to provide 7-aminocephalosporanic acid on a more economic basis than the chemical process currently in use.
However, this search for a successful microbiological process has largely proved futile, certainly with respect to one of commercial scale. This is a result of the particular characteristics of the aminoadipoyl side chain of the cephalosporin C molecule, since, by contrast, penicillin G, which has a phenylacetyl side chain, has been successfully deacylated by enzymatic cleavage using penicillin acylase produced by a variety of microorganisms. Reports of successful one-step enzymatic deacylation of cephalosporin C in the literature, on the other hand, are often unreproducible or provide only very marginal yields.
Moreover, no person to date has succeeded in isolating and sequencing an enzyme from the genus Bacillus, cephalosporin C amidase, which can achieve one-step cleavage of the aminoadipoyl side chain of cephalosporin C. Nor, has anyone isolated and sequenced the gene which encodes the cephalosporin C amidase enzyme, or succeeded in expressing that gene in a prokaryotic host.
A summary of the literature which describes these ongoing efforts to achieve enzymatic cleavage of cephalosporin C is set out below.
______________________________________ 1. One-Step Enzymatic Deacylation: Ceph C .fwdarw. 7-ACA Dev. Ind. Microbiol., 5, 349 (1964) Achromobacter, U.S. Pat. No. 3,239,394 Brevibacterium, (Merck) Flavobacterium Soil enrichment method of screening and selecting for microorganisms Jap. Pat. Pub. 53-94093 (1978) Pseudomonas sp. BN-188 (Meiji) Jap. Pat. Pub. 52-143289 (1977) Aspergillus sp. U.S. Pat. No. 4,141,790 Alternaria sp. (Meiji) U.S. Pat. No. 4,774,179 (1988) Pseudomonas Jap. Pat. Pub. 61-21097 (1986) sp. SE-83 and SE-495 Jap. Pat. Pub. 61-152286 (1986) (Asahi) Fr. Pat. 2,241,557 (1975) Bacillus cereus (Aries) var. fluorescens Jap. Pat. Pub. 52-082791 (1977) Bacillus megaterium (Toyo Jozo) NRRL B 5385 N-(N'-phenylthiocarbamyl)- cephalosporin C .fwdarw. 7-ACA Ger. Pat. 3,447,023 (1986) Bacillus licheniformis (Hoechst) In the presence of .alpha.-keto acids, enzyme is D-amino acid transaminase EP-A- 0 321 849 Pseudomonas, (Hoechst) Bacillus subtilis, Anthrobacter parafineus .gamma.-glutamyl transpeptidase 2. One-Step Enzymatic Deacylation: Penicillin G .fwdarw. 6-APA Jap. Pat. Pub. 58-190399 (1983) Bacillus megaterium (Shionogi) var. penicilliticum ATCC 14945 U.S. Pat. No. 3,144,395 (1964) Bacillus megaterium (Olin Mathieson) var. penicilliticum ATCC 14945 Br. Pat. Pub. 2,142,336A (1985) Bacillus megaterium (Squibb) Applied and Environmental (1988) Anthrobacter viscosus Microbiology, 54, 2603-2607 E. coli (Banyu) Pseudomonas Penicillin acylase .alpha.-subunit specificity genetic sequence 3. Two-Step Enzymatic Deacylation: Ceph C .fwdarw. 7-ACA U.S. Pat. No. 3,960,662 (1976) Pseudomonos sp. Agric. Biol. Chem. 45, 1561-67 (1981) (Toyo Jozo) Deamination with D-amino acid oxidase followed by deacylation EP-A- 0 275 901-A2(1988) (Hoechst) i) Ceph C .fwdarw. GL-7-ACA* Trigonopsis variabilis [U.S. Pat. No. 3,801,458 (1974) (Glaxo)] ii) Gl-7-ACA* .fwdarw. 7-ACA Pseudomonas gamma-glutamyl- Anthrobacter parafineus transpeptidase Bacillus subtilis 4. Enzymatic Deacylation: Gl-7-ACA* .fwdarw. 7-ACA Jap. Pat. Pub. 52-128293 (1977) Bacillus, 53-86094 (1978) Anthrobacter, (Banyu) Alcaligenes 5. Enzymatic Deacylation: Other .fwdarw. 7-ACA a) Phenoxy- and Phenylacetyl 7-ADCA .fwdarw. 7-ADCA U.S. Pat. No. 3,821,081 (1974) Bacillus megaterium Process Biochem., 11, 21 (1976) (Toyo Jozo) U.S. Pat. No. 3,749,641 (1973) 61 different (Takeda) genera U.S. Pat. No. 3,915,798 (1975) Anthrobacter simplex Belg. Pat. No. 780,676 Kluyvera citrophila (Toyo Jozo) Proteus rettgeri Bacillus megaterium b) Phenoxy-7-ADCA .fwdarw. 7-ADCA U.S. Pat. No. 3,880,713 (1975) Erwinia aroideae (Glaxo) c) Cephalothin .fwdarw. 7-ACA U.S. Pat. No. 3,522,250 (1970) Escherichia coli (American Home Products) d) Various cephalosporins .fwdarw. 7-ACA U.S. Pat. No. 3,930,949 (1976) E. coli (Bayer) penicillin acylase U.S. Pat. No. 3,962,036 (1976) E. coli, (Ciba-Geigy) Bacillus megaterium, subtilis, Micrococcus roseus, lysodeikticus 3-lower alkoxy-7-acyl Alcaligenes faecalis, cephalosporins; microorganisms Aerobacter cloacae, possessing acylase activity Fusarium avenaceum, semitectum, Emericellopsis minima, Penicillium chrysogenum, Aspergillus ochraceus, Trichophyton mentagrophytes, Epidermophyton floccosum, Streptomyces lavendulae Jap. Pat. Pub. 50-107186 (1975) Anthrobacter, (Toyo Brewing) Bacillus, Escherichia, phenylacetamido 7-ACA Kluyvera, derivatives are Micrococcus, deacylated Nocardia, Proteus, Xanthomonas 6. Enzymatic Acylation: 7-ACA .fwdarw. Other U.S. Pat. No. 3,945,888 (1976) E. coli, (Takeda) Bacillus, Proteus, 7-ACA .fwdarw. cephalosporins Pseudomonas Jap. Pat. Pub. 54-110394 (1979) Anthrobacter viscosus (Banyu) 7-ACA .fwdarw. cephapirin 7. One-Step-Recombinant: Ceph C .fwdarw. 7-ACA Jap. Pat. Pub. 60-110292 (1985) Comamonas (Asahi) Recombinant E. coli with gene from Comamonas sp. SY-77-1; one-step conversion Jap. Pat. Pub. 61-152-286 (1986) Pseudomonas (Asahi) Recombinant E. coli with gene from Pseudomonas sp. SE83; genetic sequences described and claimed one step process already claimed in U.S. Pat. No. 4,774,179 Jap. Pat. Pub. 63-74488 (1988) Trigonopsis variabilis, (Asahi) Comamonas Recombinant E. coli expression of D-amino acid oxidase and GL-7-ACA* acylase construct ______________________________________ GL-7-ACA = glutaryl 7ACA = 3acetoxymethyl-7-.beta.-(4-carboxybutanamido)ceph-3-em-4-carboxylic acid.